We update the proof that the sign recognition particle (SRP) recognizes sign sequences through the NG area in the SRP54 protein subunit. Using a recently developed crosslinking methodology (Fancy, DA and Kodadek, T. (1999) Proc. Natl. Acad. Sci. USA 96, 6020-6024; Correction (1999) Proc. Natl. Acad. Sci. USA 96, 1317 ), we found that the sign peptides cross-link the SRP protein of Escherichia coli Ffh (the homologue of the mammalian SRP54 subunit) through the NG area.
Within the NG area, the cross-linking website is mapped to the ras-like C-terminal subdomain called area G. This consequence is distinguished from previous research, which concluded based primarily on the cross-linking of nascent chains that the sequence of signs is true for the contiguous area M. As unbiased evidence for a direct binding interaction between the NG area and the sign sequence, we found that the NG area of Ffh binds to the sign peptides as a remote entity. Our results recommend that the NG area write a considerable part of the binding website for the sign sequence.
P53-regulated networks of protein, mRNA, miRNA and lncRNA expression revealed by labeling of pulsed safe isotopes integrated with amino acids in cellular tradition (pSILAC) and later era sequencing analysis (NGS).
We decided the impact of p53 activation on de novo protein synthesis using quantitative proteomics (safe isotope labeling pulsed with amino acids in the cellular tradition / pSILAC) within the SW480 cell line of most colorectal cancers. This was mixed with expression analysis of mRNA and antisense RNA by subsequent technology sequencing (RNA-, miR-Seq). Furthermore, the DNA binding of the entire p53 genome was analyzed by chromatin immunoprecipitation (ChIP-Seq).
Thus, we recognized differentially regulated proteins (542 up, 569 down), mRNA (1258 up, 415 down), miRNA (111 up, 95 down) and lncRNA (270 up, 123 down). Modifications in the protein and mRNA expression ranges confirmed a constructive correlation (r = 0.50, p <0.0001). In total, we detected 133 direct p53 target genes that had been differentially expressed and exhibited p53 occupation in the vicinity of its promoter.
The extra transcriptionally induced genes showed occupied p53 binding web sites (4.3% mRNA, 7.2% miRNA, 6.3% lncRNA, 5.9% proteins) than the repressed genes (2.4 % mRNA, 3.2% miRNA, 0.8% lncRNA, 1.9% protein), suggesting oblique mechanisms. of repression. About 50% of the negatively regulated proteins showed p53-induced miRNA seed matching sequences within the corresponding 3'-UTRs.
Furthermore, the proteins repressed by p53 overlapped considerably with these which were shown beforehand to be repressed by miR-34a. We confirmed the up-regulation of the new direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and confirmed that the ectopic expression of LINC01021 inhibits proliferation in SW480 cells. Furthermore, the mRNAs of KLF12, HMGB1 and CIT had been confirmed as direct targets of the miR-34a, miR-205 and miR-486-5p induced by p53, respectively.
Mapping of the signal sequence binding site in SRP reveals a significant role for the NG domain.
Consistent with the lack of p53 operation throughout tumor development, elevated expression of KLF12, HMGB1, and CIT was detected in the higher stages of most cancers. In conclusion, the combination of a series of omic strategies allowed the excellent identification of p53 direct and oblique effectors that present new insights and lead to p53-mediated tumor suppression mechanisms.
Post-Generation Sequencing (NGS) for High Quality Microbial Water Assessment: Current Progress, Challenges, and Future Alternatives.
High water quality is an emerging property of an elegant system composed of interacting microbial populations and released microbial and chemical contaminants. Research leveraging applied next-generation sequencing (NGS) sciences is offering new insights into the ecology of microbe-mediated processes affecting contemporary high water quality reminiscent of algal blooms, biodegradation of contaminants, and shedding. of pathogens. In addition, sequencing strategies that focus on hypervariable areas of small subunit rRNA (SSU) have allowed the identification of characteristic microbial species that function as bioindicators of wastewater contamination in these settings.
Amplicon sequencing in the past, metagenomic and metatrans analyzes